Cartilage defect regeneration by ex vivo engineered autologous microtissue--preliminary results.

We analysed the improvement of cartilage defect regeneration by the use of microspheres of autologous chondrocytes. Autologous chondrocytes from minipigs were cultured using the microsphere technology. Cartilage defects were surgically introduced to tibia-femoral joints. Tissue constructs were then implanted into defect sites. Histological, immunohistological and transmission electron microscopic analyses were performed. Histological and ultrastructural investigations of chondrospheres revealed a cartilage-like tissue formation, indicated by phenotypically differentiated chondrocyte-like cells surrounded by de novo synthesised chondrogenic extracellular matrix. Clinical inspection of defects demonstrated nearly complete cartilage regeneration in the microtissue treated defect sites, whereas controls exhibited irregular fibrous tissue formation. In vitro-expanded articular chondrocytes are able to form a microtissue capable of repairing cartilage defects in vivo, improving regeneration of cartilage defects.

Cyclooxygenases (COX-1 and COX-2) for tissue engineering of articular cartilage--from a developmental model to first results of tissue and scaffold expression.

Tissue engineering of articular cartilage remains an ongoing challenge. Since tissue regeneration recapitulates ontogenetic processes the growth plate can be regarded as an innovative model to target suitable signalling molecules and growth factors for the tissue engineering of cartilage. In the present study we analysed the expression of cyclooxygenases (COX) in a short-term chondrocyte culture in gelatin-based scaffolds and in articular cartilage of rats and compared it with that in the growth plate. Our results demonstrate the strong cellular expression of COX-1 but only a focal weak expression of COX-2 in the seeded scaffolds. Articular cartilage of rats expresses homogeneously COX-1 and COX-2 with the exception of the apical cell layer. Our findings indicate a functional role of COX in the metabolism of articular chondrocytes. The expression of COX in articular cartilage and in the seeded scaffolds opens interesting perspectives to improve the proliferation and differentiation of chondrocytes in scaffold materials by addition of specific receptor ligands of the COX system.

Clinical experience in cell-based therapeutics: intervention and outcome.

Disc herniation treated by discectomy results in a significant loss of nucleus material and disc height. Biological restoration through the use of autologous disc chondrocyte transplantation (ADCT) offers a potential to achieve functional integration of disc metabolism and mechanics. Nucleus regeneration using autologous cultured disc-derived chondrocytes has been demonstrated in a canine model and in clinical pilot studies. In 2002 a prospective, controlled, randomized, multicentre study comparing safety and efficacy of ADCT plus discectomy, with discectomy alone was initiated. The clinical goals were to provide long-term pain relief, maintain disc height, and prevent adjacent segment disease. Interim analysis was performed after 2 years; Oswestry (Low Back Pain/disability), Quebec Back Pain Disability Scale, as well as Prolo and VAS Score were used for the evaluation. Disc height was assessed by MRI. A clinically significant reduction of low back pain in the ADCT-treated group was shown by all three pain score systems. The median total Oswestry Score was 2 in the ADCT group compared with 6 in the control group. Decreases in the Disability index in ADCT-treated patients correlated with the reduction of low back pain. Decreases in disc height over time were only found in the control group, and of potential significance, intervertebral discs in adjacent segments appeared to retain hydration when compared to those adjacent to levels that had undergone discectomy without cell intervention.

Translation from research to applications.

The article summarizes the collective views expressed at the fourth session of the workshop Tissue Engineering--the Next Generation, which was devoted to the translation of results of tissue engineering research into applications. Ernst Hunziker described the paradigm of a dual translational approach, and argued that tissue engineering should be guided by the dimensions and physiological setting of the bodily compartment to be repaired. Myron Spector discussed collagen-glycosaminoglycan (GAG) scaffolds for musculoskeletal tissue engineering. Jeanette Libera focused on the biological and clinical aspects of cartilage tissue engineering, and described a completely autologous procedure for engineering cartilage using the patient's own chondrocytes and blood serum. Arthur Gertzman reviewed the applications of allograft tissues in orthopedic surgery, and outlined the potential of allograft tissues as models for biological and medical studies. Savio Woo discussed a list of functional tissue engineering approaches designed to restore the biochemical and biomechanical properties of injured ligaments and tendons to be closer to that of the normal tissues. Specific examples of using biological scaffolds that have chemoattractants as well as growth factors with unique contact guidance properties to improve their healing process were shown. Anthony Ratcliffe discussed the translation of the results of research into products that are profitable and meet regulatory requirements. Michael Lysaght challenged the proposition that commercial and clinical failures of early tissue engineering products demonstrate a need for more focus on basic research. Arthur Coury described the evolution of tissue engineering products based on the example of Genzyme, and how various definitions of success and failure can affect perceptions and policies relative to the status and advancement of the field of tissue engineering.

Disc chondrocyte transplantation in a canine model: a treatment for degenerated or damaged intervertebral disc.

STUDY DESIGN: Disc degeneration and osteoarthritis are diseases of the matrix. Chondrocytes that have been removed from damaged cartilaginous tissues maintain a capacity to proliferate, produce, and secrete matrix components, and respond to physical stimuli such as dynamic loading. A dog model was used to investigate the hypothesis that autologous disc chondrocytes can be used to repair damaged intervertebral disc. OBJECTIVES: Given the capacity for the cells in vitro to produce matrix molecules that would be appropriate for disc chondrocytes, the focus of the experiment was to investigate whether the cells would continue to sustain metabolic function after transplantation. SUMMARY OF THE BACKGROUND DATA: No evidence for long-term integration exists for cell transplantation in species other than rats and rabbits. Furthermore, no controlled studies of 1-year duration have been published. MATERIALS AND METHODS: Disc chondrocytes were harvested and expanded in culture under controlled and defined conditions, returned to the same animals from which they had been sampled (autologous transplantation) via percutaneous delivery. The animals were analyzed at specific times after transplantation by several methods to examine whether disc chondrocytes integrated with the surrounding tissue, produced the appropriate intervertebral disc extracellular matrix, and might provide a formative solution to disc repair. RESULTS: In the context of degenerative changes in an injury model: (1) autologous disc chondrocytes were expanded in culture and returned to the disc by a minimally invasive procedure after 12 weeks; (2) disc chondrocytes remained viable after transplantation as shown by Bromodeoxyuridine incorporation and maintained a capacity for proliferation after transplantation as depicted by histology; (3) transplanted disc chondrocytes produced an extracellular matrix that displayed composition similar to normal intervertebral disc tissue. Positive evidence of proteoglycan content was supported by accepted histochemical staining techniques such as Safranin O-Fast Green; (4) both type II and type I collagens were demonstrated in the regenerated intervertebral disc matrix by immunohistochemistry after chondrocyte transplantation; and (5) when the disc heights were analyzed for variance according to treatment, a statistically significantcorrelation between transplanting cells and retention of disc height was achieved. CONCLUSIONS: Autologous chondrocyte transplantation is technically feasible and biologically relevant to repairing disc damage and retarding disc degeneration.

In vitro engineering of human autogenous cartilage.

A challenge in tissue engineering is the in vitro generation of human cartilage. To meet standards for in vitro-engineered cartilage, such as prevention of immune response and structural as well as functional integration to surrounding tissue, we established a three-dimensional cell culture system without adding exogenous growth factors or scaffolds. Human chondrocytes were cultured as spheroids. Tissue morphology and protein expression was analyzed using histological and immunohistochemical investigations on spheroid cryosections. A cartilage-like tissue similar to naturally occurring cartilage was generated when spheroids were cultured in medium supplemented only with human serum. This in vitro tissue was characterized by the synthesis of the hyaline-specific proteins collagen type II and S-100, as well as the synthesis of hyaline-specific mucopolysaccharides that increased with prolonged culture time. After 3 months, cell number in the interior of in vitro tissues was diminished and was only twice as much as in native cartilage. Additionally, spheroids quickly adhered to and migrated on glass slides and on human condyle cartilage. The addition of antibiotics to autologous spheroid cultures inhibited the synthesis of matrix proteins. Remarkably, replacing human serum by fetal calf serum resulted in the destruction of the inner part of the spheroids and only a viable rim of cells remained on the surface. These results show that the spheroid culture allows for the first time the autogenous in vitro engineering of human cartilage-like tissue where medium supplements were restricted to human serum.